Non-Canonical Notch Regulation of Cardiovascular Progenitors

描述（由申请人提供）：心血管祖细胞（CPC）由于其独特的扩增和分化成各种心脏细胞类型的能力，在心脏再生医学方面具有巨大的治疗潜力。然而，为了利用再生治疗，我们需要了解CPC自我更新和谱系特异性分化的机制。目前的建议集中在阐明一个新的作用Notch信号在CPC的维持和分化。Notch是一种进化上保守的跨膜蛋白，在许多细胞命运决定中起关键作用。典型的Notch信号传导是通过细胞外配体与Notch的结合来启动的。这导致Notch的细胞内切割和易位进入细胞核，在那里它结合到转录介质RBP-J用于基因激活。我证明了Notch 1缺陷型CPC随着增殖的增加而显著扩增，类似于用活性2-连环蛋白刺激的CPC的表型。在RBP-J缺陷的CPC中未观察到这种表型，表明Notch的非典型作用。Notch 1缺陷显著增加2-连环蛋白信号传导。这种增加不是由2-连环蛋白mRNA的上调介导的，而是由活性2-连环蛋白蛋白的积累介导的，表明了翻译后调节。有趣的是，2-连环蛋白的Notch调节不需要Notch的经典配体依赖性膜切割或2-连环蛋白指导的蛋白酶体降解，但它确实需要将膜Notch运输到溶酶体的内吞蛋白。此外，膜结合的Notch，通常被认为是生物惰性的，与活性2-连环蛋白物理相关，并抑制活性2-连环蛋白的积累。这些发现揭示了膜Notch在调节活性2-Catenin蛋白水平中的先前未描述的作用，并为探索这种作用在CPC的维持和分化中的机制奠定了基础。我建议测试的假设，Notch拮抗CPC自我更新/扩张的溶酶体降解活性2-连环蛋白在配体/转录独立的方式。本发明的具体目的是（1）确定Notch 1缺陷型CPC是否比分化更有利于自我更新，以及这种作用是否需要2-连环蛋白;（2）测试膜结合Notch 1是否影响CPC扩增和分化，以及细胞事件是否需要2-连环蛋白;（3）确定溶酶体活性在膜结合Notch和活性2-Catenin降解之间的联系中的作用。所提出的工作将为理解控制CPC自我更新/分化决定的机制提供基本见解，这是CPC介导的心脏再生疗法的先决条件。膜Notch的生物学在CPC领域以及干细胞中完全未被探索。鉴于Notch和Wnt/2-Catenin信号在几乎所有已知的干/祖细胞命运决定中的高度保守作用，这些研究将为涉及干/祖细胞的再生医学研究开辟新的途径。 公共卫生相关性：了解发育成心脏的细胞的生物学，称为多能心血管祖细胞（CPC），是实现未来细胞介导的心脏治疗的关键。这项研究旨在阐明一种名为Notch的蛋白质在CPC自我更新和分化中的配体/转录独立作用。我们的工作将为理解Notch调控的多能CPC的自我更新-分化过程提供机制上的见解，这将促进未来基于细胞的心脏治疗，并为非经典Notch生物学的研究开辟新的途径。

DESCRIPTION (provided by applicant): Cardiovascular progenitor cells (CPCs) hold tremendous therapeutic potential for cardiac regenerative medicine due to their unique ability to expand and to differentiate into various heart cell types. However, to take advantage of regenerative therapy, we need to understand the mechanisms underlying the self-renewal and lineage-specific differentiation of CPCs. The current proposal focuses on elucidating a novel role of Notch signaling in CPC maintenance and differentiation. Notch is an evolutionarily conserved transmembrane protein that plays critical roles in numerous cell-fate decisions. Canonical Notch signaling is initiated by binding of extracellular ligands to Notch. This leads to intracellular cleavage and translocation of Notch into the nucleus where it binds to the transcriptional mediator RBP-J for gene activation. I demonstrated that Notch1-deficient CPCs expand dramatically with increased proliferation, similar to the phenotype of CPCs stimulated with active 2-Catenin. This phenotype is not observed in CPCs deficient for RBP-J, suggesting a non-canonical role of Notch. Notch1-deficiency significantly increased 2-Catenin signaling. This increase was not mediated by upregulation of 2-Catenin mRNA but rather by accumulation of active 2-Catenin protein, suggesting post- translational regulation. Intriguingly, the Notch regulation of 2-Catenin protein did not require classical ligand- dependent membrane cleavage of Notch or the 2-Catenin directed proteasomal degradation, but it did require endocytic proteins that traffic membranous Notch to the lysosome. Moreover, membrane-bound Notch, conventionally considered biologically inert, physically associated with active 2-Catenin and inhibited accumulation of active 2-Catenin protein. These findings reveal a previously undescribed role of membrane Notch in regulating active 2-Catenin protein levels and set the stage for a mechanistic exploration of this role in the maintenance and differentiation of CPCs. I propose to test the hypothesis that Notch antagonizes CPC self-renewal/expansion by lysosomal degradation of active 2-Catenin in a ligand/transcription-independent fashion. The specific aims of this proposal are (1) To determine if Notch1-deficient CPCs favor self-renewal over differentiation and if 2-Catenin is required for this effect; (2) To test whether membrane-bound Notch1 affects CPC expansion and differentiation and whether the cellular events require 2-Catenin; (3) To identify the role of lysosomal activity in the link between membrane-bound Notch and active 2-Catenin degradation. The proposed work will provide fundamental insights into the understanding of mechanisms controlling CPC self- renewal/differentiation decisions, a prerequisite for CPC-mediated cardiac regenerative therapeutics. The biology of membrane Notch is completely unexplored in the field of CPCs as well as in stem cells. Given highly conserved roles of Notch and Wnt/2-Catenin signaling in nearly all known stem/progenitor cell fate decisions, these studies will open up new avenues of research for regenerative medicine involving stem/progenitor cells. PUBLIC HEALTH RELEVANCE: Understanding the biology of the cells that develop into the heart, called multipotent cardiovascular progenitor cells (CPCs), is key to realizing the promise of future cell-mediated cardiac therapeutics. The proposed research aims to elucidate the ligand/transcription-independent role of a protein called Notch in CPC self- renewal and differentiation. Our work will provide mechanistic insights into understanding the self-renewal- differentiation processes of multipotent CPCs regulated by Notch, which will facilitate future cell-based cardiac therapeutics and open new avenues of investigation for non-canonical Notch biology.